Field of the Invention
The present invention relates to methods for co-silencing expression of genes in a filamentous fungal strain. The present invention also relates to methods for identifying genes encoding biological substances of interest in a filamentous fungal cell using such co-silencing methods.
Description of the Related Art
Filamentous fungal strains are widely used for the production of biological substances of commercial value. However, filamentous fungal strains with desirable traits of expression and secretion of a biological substance may not necessarily have the most desirable characteristics for successful fermentation. The production of a biological substance may be accompanied by the production of other substances, e.g., enzymes that degrade the biological substance or co-purify with the biological substance, which can complicate use of the biological substance.
One solution to these problems is to inactivate the gene(s) involved in the production of the undesirable substance. Inactivation can be accomplished by deleting or disrupting the gene(s) using methods well known in the art. However, in some cases, inactivation of the gene may be difficult because of poor targeting to homologous regions of the genome. Inactivation can also be accomplished by random mutagenesis, which is not always specific for the intended target gene and other mutations are often introduced into the host organism. In other situations, the gene and its product may be required for survival of the filamentous fungal strain. Where multiple genes are to be inactivated by deletion or disruption, the task can be very cumbersome and time-consuming. Furthermore, when highly homologous members of gene families exist, deletion or disruption of all members can be very tedious and difficult.
In recent years various forms of epigenetic gene regulation have been described (Selker, 1997, Trends Genet. 13: 296-301; Matzke and Matzke, 1998, Cell. Mol. Life. Sci. 54: 94-103). These processes influence gene expression by modulating the levels of messenger RNA (Hammond and Baulcombe, 1996, Plant Mol. Biol. 32: 79-88; Xi-song Ke et al., 2003, Current Opinion in Chemical Biology 7: 516-523) via micro RNAs (Morel et al., 2000, Curr. Biol. 10: 1591-1594; Bailis and Forsburg, 2002, Genome Biol. 3, Reviews 1035; Grewal and Moazed, 2003, Science 301: 798-802).
Based on genetic studies of Drosophila melanogaster and Caenorhabditis elegans, RNA interference (RNAi), also known as post-transcriptional gene silencing (in plants), is understood to involve silencing expression of a gene by assembly of a protein-RNA effector nuclease complex that targets homologous RNAs for degradation (Hannon, 2002, Nature 418: 244-251). The processing of double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) is accomplished by a family of enzymes known as Dicer (Bernstein et al., 2001, Nature 409: 363). Dicer, a member of the RNase III family of endonucleases that specifically cleaves dsRNA, is responsible for digestion of dsRNA into siRNAs ranging from 20-25 nucleotides (Elbashir et al., 2001, Nature 411: 494). These siRNAs then associate with the RNA Induced Silencing Complex (RISC) (Elbashir et al., 2001, Genes and Dev. 15: 188; NyKanen et al., 2001, Cell 197: 300; Hammond et al., 2001, Science 293: 1146). Although not well understood, RISC targets the mRNA from which the anti-sense fragment is derived followed by endo and exonuclease digestion of the mRNA effectively silencing expression of that gene. RNAi has been demonstrated in plants, nematodes, insects, mammals, and filamentous fungi (Matzke and Matzke, 1998, supra; Kennerdell et al., 2000, Nat. Biotechnol. 18: 896-8; Bosher et al., 1999, Genetics 153: 1245-56; Voorhoeve and Agami, 2003, Trends Biotechnol. 21: 2-4; McCaffrey et al., 2003, Nat. Biotechnol. 21: 639-44; WO 03/050288; WO 01/49844; WO 98/53083; and WO 05/056772).
Transitive RNAi refers to the movement of the silencing signal beyond a particular gene. In plants, transitive silencing has been found to occur both upstream and downstream of the mRNA targeted for gene silencing by double-stranded RNA (Fabian et al., 2002, Plant Cell 14: 857-867; Garcia-Perez et al., 2004, The Plant Journal 38: 594-602; Vaistij et al., 2002, The Plant Cell 14: 857-867; Van Houdt et al., 2003, Plant Physiol. 131: 245-253). In Caenorhabditis elegans, transitive RNAi has been described as silencing of the transcript upstream of the dsRNA inducer (Alder et al., 2003, RNA J. 9: 25-32; Hannon, 2002, Nature 418: 244-251; Sijen et al., 2001, Cell 107: 465-476). In C. elegans, descriptions of transitive RNAi indicate that in addition to siRNAs derived from the dsRNA inducer, secondary siRNAs sharing homology with 5′ flanking sequences are generated, presumably the result of RNA-dependent RNA polymerase (RdRP) and Dicer activity (Bleys et al., 2006, RNA J. 12: 1633-1639; Petersen et al., 2005, Plant Molecular Biology 58: 575-583). Transitive RNAi is not ubiquitous among insects and mammals (Chi et al., 2003, Proc. Natl. Acad. Sci. USA 100: 6343-6346; Hoa et al., 2003, Insect Biochemistry and Molecular Biology 33: 949-957; Roignamt et al., 2003, RNA J. 9: 299-308).
Transitive RNAi differs from conventional RNAi. Although double-stranded RNA serves as the inducer of both RNAi and transitive RNAi, transitive RNAi appears to require an RdRP, whereas RNAi alone does not. Consequently, in organisms demonstrating transitive RNAi, gene silencing is not limited by the boundaries of double-stranded RNA, and gene silencing can extend into flanking sequences. However, in organisms lacking transitive RNAi, gene silencing is confined within the region of double strandedness. RNA interference by both conventional and transitive mechanisms can give rise to strains in which expression of the target gene is either partially or completely suppressed (Brody and Maiyuran, 2009, Industr. Biotechnol. 5: 53-60; Fernandez et al., 2012, Fungal Genet. Biol. 49: 294-301).
It would be an advantage in the art to have alternative methods for silencing expression of more than one gene for strain development and improvement, functional genomics, and pathway engineering of filamentous fungal strains. It would also be an advantage in the art to overcome one of the limitations of previous RNAi methods in filamentous fungi, i.e., how to identify strains/transformants in which gene silencing is most effective.
The present invention relates to methods for silencing expression of more than one gene in filamentous fungal strains and methods of using silencing of phenotypic markers to identify transformants in which silencing of a target gene is most efficient.